US 2003/0168392 A1 discloses a multi-dimensional liquid chromatograph separation system. The analytes are separated on a first analysis system consisting of a first column and a first mobile phase, and are trapped on trapping columns. The trapped analytes are subsequently loaded onto the second analysis system consisting of a second column and a second mobile phase. The document discloses the trapping and loading mechanism consisting of a combination of switching valves necessary to produce the serial separations.
An overview on the purification of proteins using magnetic adsorbent particles is given by Franzreb M., et al. Appl. Microbiol. Biotechnol. 70 (2006) 505-516. Particularly, Hubbuch, J. J., et al. Biotechnol Bioeng 79 (2002) 301-313 describe a high-gradient magnetic separation system comprising a filter chamber filled with woven wire mesh of steel, whereby the filter chamber is positioned between the plane pole shoes of an electromagnet which can be switched on or off. The filter chamber comprises two openings at opposite ends, at the top and the bottom of the chamber. The two openings of the filter chamber are fluidically connected and a pump is integrated in the fluidic connection. Thereby a loop is formed such that a liquid phase can be cycled within the loop. Between the pump and the bottom opening of the chamber the authors disclose a three way valve, suitable for fluidically connecting the filter chamber with a batch reactor and a first buffer reservoir. Between the pump and the top opening of the filter chamber a four way valve is disclosed, the valve being suitable for making a fluidic connection with a second or a third buffer reservoir, or a fraction collector. The authors further disclose the use of such a fluidic system for purifying trypsin from crude pancreatin. To this end, magnetic particles functionalized with benzamidine are incubated in the batch reactor with a suspension of crude pancreatin, whereby trypsin is adsorbed to the particles. The suspension with the magnetic particles is fed into the loop together with binding buffer. The suspension is circulated in the loop and passed through the filter chamber several times with the electromagnet being switched on, whereby the magnetic particles are immobilized in the filter chamber. Subsequently, the liquid phase is exchanged by a washing buffer, the immobilized magnetic particles are released by switching the magnet off, and circulated in the loop with the washing buffer. During the washing step trypsin remains adsorbed to the magnetic particles. After a further immobilization step, the washing buffer is substituted with an elution buffer and elution is performed by again releasing the magnetic particles and circulating buffer and particles in the loop. The last step is the recovery of the elution buffer with trypsin from the system while the magnetic particles are retained in the filter chamber.
WO 2007/009519 discloses a system in which the separation chamber comprises, between two fits, a fluidic-bed of functionalized magnetic particles. A magnetic field can be applied and as a result the magnetic particles are immobilized at the walls of the separation chamber. A liquid phase containing an analyte is circulated through the separation chamber. Washing and elution steps can be performed.
US 2008/0217254 discloses a magnetic trap in a fluid handling system additionally comprising a plurality of rotary valve switching units. The magnetic trap comprises a plurality of pairs of magnets. A pair comprises two magnets which are placed side by side in opposite orientation, generating a magnetic field gradient. Several such pairs of magnets are positioned on a rotary device. A tube being part of the fluid system is positioned in a circular arrangement and in close proximity to the magnets such that high magnetic field gradients are applied to the lumen of the tube. When the device rotates the magnets, the magnetic field gradients are moved along the tube lumen. The device can be rotated clockwise or counterclockwise. Magnetic beads are trapped in the tube lumen. Movement of magnetic beads in the tube lumen of the trap is controlled by the movement of the magnetic field gradients.
For the majority of in-vitro diagnostic analyses it is necessary to extract one or more target analytes from complex sample materials (serum, plasma, whole blood, urine etc.). In this connection the respective target analytes are concentrated by different processes whereas components of the sample matrix which would hinder the subsequent analysis (e.g. proteins, peptides, salts) are depleted. The following extraction methods have been previously used to concentrate target analytes: protein precipitation with organic solvents or acids; liquid-liquid extraction (solvent extraction) with an evaporation step; solid phase extraction on cartridges which contain particles with defined surface structures (especially hydrocarbon-functionalized silica particles; solid phase extraction, SPE).
Extraction methods known from the prior art require a large amount of manual work. Methods for automating such extraction processes have up to now been technically very elaborate and have numerous disadvantages. They require, on the one hand, very demanding mechanical constructions e.g. pipetting systems and/or vacuum systems. On the other hand, the known methods require a large amount of solid and liquid consumables e.g. extraction cartridges, extraction plates, solvents. Furthermore, they require a long processing period and are characterized by a low sample throughput and limited series lengths.
An important aim of the work towards the present invention was to develop an extraction system which, in combination with a separation and analytical system, forms a substantially closed system. The liquid system according to the invention which is preferably a high pressure liquid system overcomes the limitations of current automated extraction processes. A particular object of the invention was to overcome and simplify certain disadvantages concerning the manipulation of magnetic or paramagnetic particles during the extraction process.